Method for Selectively Inhibiting the Activity of ACAT1 in the Treatment of Alzheimer&#39;s Disease

ABSTRACT

The present invention features methods for decreasing the size and density of amyloid plaques, decreasing cognitive decline associated with amyloid pathology, and treating Alzheimer&#39;s disease by selectively inhibiting the activity of Acyl-CoA:Cholesterol Acyltransferase 1, but not Acyl-CoA:Cholesterol Acyltransferase 2.

INTRODUCTION

This application is a continuation-in-part application of U.S.application Ser. No. 13/072,915, filed Mar. 28, 2011, which is acontinuation-in-part application of PCT/US2009/056601, filed Sep. 11,2009, which claims the benefit of priority of U.S. ProvisionalApplication No. 61/103,658, filed Oct. 8, 2008, the content of which isincorporated herein by reference in its entirety.

This invention was made with government support under grant numberR01HL060306 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Alzheimer's disease is characterized by two pathological hallmarks,namely extracellular accumulation of plaques, which are aggregates ofamyloid beta (Aβ) peptides derived from proteolytic cleavages of amyloidprecursor protein (APP), and intracellular accumulation ofhyperphosphorylated tau (Hardy & Selkoe (2002) Science 297:353-356). APPcan be cleaved via two competing pathways, the alpha and the betasecretase pathways, which are distinguished by different subcellularsites of proteolysis and cleavage points within APP (Thinakaran & Koo(2008.) J. Biol. Chem. 283:29615-29619). Several proteases are capableof producing the alpha-cleavage, after which the gamma-secretase complexthat includes presenilin 1 as a catalytic subunit, further cleaves theAPP fragment to produce small, non-amyloidogenic fragments. Thebeta-secretase pathway involves sequential cleavages by beta-secretaseand gamma-secretase complexes, and generates Aβ. APP and secretases areall membrane bound proteins/enzymes. Studies have shown that cholesterolcontent in cells can affect the production of Aβ, in part by the abilityof cholesterol to modulate the enzyme activities of various secretasesin cell membranes (Wolozin (2004) Neuron 41:7-10). Cholesterolmetabolism has also been implicated in the pathogenesis of Alzheimer'sdisease in other manners (Jiang, et al. (2008) Neuron 58:681-693;Wellington (2004) Clin. Genet. 66:1-16; Hartmann (2001) Trends Neurosci.24:S45-48).

In the brain, cholesterol is derived from endogenous biosynthesis(Dietschy & Turley (2004) J. Lipid Res. 45:1375-1397). The transcription‘factor SREBP2 controls the expression of enzymes involved incholesterol biosynthesis, including the rate-limiting enzyme HMG-CoAreductase (HMGR) (Goldstein, et al. (2006) Cell 124:35-46). Othertranscription factors, including liver X receptors (LXRs), control theexpression of proteins which function in cholesterol transport (Repa &Mangelsdorf (2000) Annu. Rev. Cell Dev. Biol. 16:459-481; Beaven &Tontonoz (2006) Annu. Rev. Med. 57:313-329), including apoE, ABCA1, andothers (Wang, et al. (2008) FASEB J. 22:1073-1082; Tarr & Edwards (2008)J. Lipid Res. 49:169-182). In the brain, cholesterol can beenzymatically converted by a brain-specific enzyme, 24-hydroxylase(CYP46A1) (Russell, et al. (2009) Annu. Rev. Biochem. 78:1017-1040), toan oxysterol called 24S-hydoxycholesterol (2450H); the concentration of24SOH far exceeds those of other oxysterols in the brain (Lutjohann, etal. (1996) Proc. Natl. Acad. Sci. USA 93:9799-9804 Bjorkhem (2006) J.Intern. Med. 260:493-508; Karu, et al. (2007) J. Lipid Res. 48:976-987).Various oxysterols, including 24SOH, can downregulate sterol synthesisin intact cells and in vitro (Song, et al. (2005) Cell Metab. 1:179-189;Wang, et al. (2008) J. Proteose Res. 7:1606-1614). When provided toneurons, 24SOH decreases the secretion of Aβ (Brown, et al. (2004) J.Biol. Chem. 279:34674-34681). However, whether 24SOH or other oxysterolscan act in similar fashion(s) in vivo remains to be demonstrated. 24SOHlevels have been shown to be decreased in brain samples from Alzheimer'sdisease patients (Heverin, et al. (2004) J. Lipid Res. 45:186-193),suggesting a relationship between 24SOH and Alzheimer's disease.

Acyl-CoA:Cholesterol Acyltransferase (ACAT) converts free cholesterol tocholesterol ester, and is one of the key enzymes in cellular cholesterolmetabolism. Two ACAT genes have been identified which encode twodifferent enzymes, ACAT1 and ACAT2 (also known as SOAT1 and SOAT2).While both ACAT1 and ACAT2 are present in the liver and intestine, thecells containing either enzyme within these tissues are distinct,suggesting that ACAT1 and ACAT2 have separate functions. Both enzymesare potential drug targets for treating dyslipidemia andatherosclerosis.

Using the non-selective ACAT inhibitor, CP-113,818 (Chang et al. (2000)J. Biol. Chem. 275:28083-28092), Alzheimer's disease-like pathology inthe brains of transgenic mice expressing human APP(751) containing theLondon (V717I) and Swedish (K670M/N671L) mutations has been demonstrated(Hutter-Paier, et al. (2004) Neuron. 44(2):227-38). Two months oftreatment with CP-113,818 was shown to reduce the accumulation ofamyloid plaques by 88%-99% and membrane/insoluble Amyloid β levels by83%-96%, while also decreasing brain cholesteryl-esters by 86%.Additionally, soluble Aβ(42) was reduced by 34% in brain homogenates.Spatial learning was slightly improved and correlated with decreased Aβlevels. In non-transgenic littermates, CP-113,818 also reducedectodomain shedding of endogenous APP in the brain.

A 50% decrease in ACAT1 expression has also been shown to reducecholesteryl ester levels by 22%, reduce proteolytic processing of APP,and decrease Aβ secretion by 40% (Huttunen, et al. (2007) FEBS Lett.581(8):1688-92) in an in vitro neuronal cell line. In this regard, ithas been suggested that ACAT inhibition could serve as a strategy totreat Alzheimer's disease (Huttunen & Kovacs (2008) Neurodegener. Dis.5(3-4):212-4).

SUMMARY OF THE INVENTION

The present invention features methods for decreasing the size anddensity of amyloid plaques, decreasing cognitive decline associated withamyloid pathology, and treating Alzheimer's Disease by administering toa subject in need of treatment an agent that selectively inhibits theexpression or activity of Acyl-CoA:Cholesterol Acyltransferase 1(ACAT1). In one embodiment, the agent has an IC₅₀ value for ACAT1 whichis at no more than one half the corresponding IC₅₀ value for ACAT2. Inanother embodiment, the agent does not inhibit the expression of ACAT2.In an alternative embodiment, the agent is a siRNA or microRNA molecule.In a further embodiment, the agent has an IC₅₀ value in the range of 1nM to 100 μM. In a particular embodiment of the invention, the agent isselectively delivered to the brain of the subject. In a specificembodiment, the agent is administered via a liposome or nanoparticle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amyloid beta 42 (Aβ1-42) levels in the hemibrains ofwild-type (WT) Alzheimer's Disease (AD) mice or AD mice lacking Acat1(Acat1^(−/−)) after injection of PBS, or injection with adeno-associatedvirus (AAV) vectors harboring a negative control miRNA or with siRNAtargeting ACAT1 (ACAT1 KD). Mice were injected at 10 months of age andanalyzed at 12 months of age. Group 1, WT mice (n=1); Group 2, mixedbackground AD mice injected with PBS (n=6); Group 3, mixed background ADmice injected with negative control AAV (n=8); Group 4, mixed backgroundAD mice injected with Acat1 KD AAV (n=13); Group 5, mixed backgroundAD/Acat1^(−/−) mice injected with PBS (n=9); Group 6, mixed backgroundAD/Acat1^(−/−) mice injected with negative control AAV (n=4); Group 7,mixed background AD/Acat1^(−/−) mice injected with Acat1 KD AAV (n=4).

DETAILED DESCRIPTION OF THE INVENTION

Amyloid beta-peptide (Abeta or Aβ) accumulation in specific brainregions is a pathological hallmark of Alzheimer's disease (AD). It hasnow been found that ACAT1, but not ACAT2, plays a significant role inamyloid pathology of AD in vivo. Specifically, ACAT1 modulates the sizesand densities of amyloid plaques and cognitive decline manifested in amouse model for the AD in vivo. Contrary to previous reports (Huttunen,et al. (2007) FEBS Lett. 581(8):1688-92), which were performed in an invitro neuronal cell line, it has now been shown that partial ACAT1deficiency leads to a significant reduction of Aβ peptide 1-42 in an invivo animal model for AD. Furthermore, in this AD mouse model, cognitivedeficits become obvious at 6 months of age; Aβ peptide 1-42 accumulationalso becomes obvious at 10 months of age. When AAV expressing the siRNAtargeting ACAT1 was delivered to the AD mice at 10 months of age, Aβ1-42 peptide reduction was demonstrated within 2 months of starting thesiRNA treatment (at 12 months of age). This result demonstrates clearlythat ACAT1 is a target for treating AD, either to treat diseaseprogression or to prevent disease from occurring.

Accordingly, the present invention features compositions and methods fordecreasing the size and density of amyloid plaques, decreasing cognitivedecline associated with amyloid pathology, and treating AD. Inaccordance with the methods of this invention, a subject having,suspected of having or predisposed to have AD is administered aneffective amount of an agent that selectively inhibits the activity ofACAT1 so that the size and density of amyloid plaques in the subject aredecreased, cognitive decline associated with amyloid pathology isdecreased, and/or the progression of the AD is slowed or prevented,thereby treating AD.

As used herein, a “selective inhibitor of ACAT1” or “ACAT1-selectiveinhibitor” is any molecular species that is an inhibitor of the ACAT1enzyme but which fails to inhibit, or inhibits to a substantially lesserdegree ACAT2. Methods for assessing the selectively of ACAT1 inhibitorsare known in the art and can be based upon any conventional assayincluding, but not limited to the determination of the half maximal(50%) inhibitory concentration (IC) of a substance (i.e., 50% IC, orIC₅₀), the binding affinity of the inhibitor (i.e., K_(i)), and/or thehalf maximal effective concentration (EC₅₀) of the inhibitor for ACAT1as compared to ACAT2. See, e.g., Lada, et al. (2004) J. Lipid Res.45:378-386 and U.S. Pat. No. 5,968,749. As one of skill in the art willunderstand, lower IC₅₀ values for an ACAT inhibitor indicates that theinhibitor is more potent or more active as an inhibitor of the activityof ACAT1 or ACAT2. Thus, a selective ACAT inhibitor is one wherein theIC₅₀ value for inhibition of ACAT1 is lower than the IC₅₀ value forinhibition of ACAT2. ACAT1 and ACAT2 proteins that can be employed insuch assays are well-known in the art and set forth, e.g., in GENBANKAccession Nos. NP_(—)000010 (human ACAT1) and NP_(—)005882 (humanACAT2). See also U.S. Pat. No. 5,834,283.

In a particular embodiment, a selective inhibitor of ACAT1 is an agentwhich has an IC₅₀ value for ACAT1 that is at least twice or, moredesirably, at least three, four, five, or six times lower than thecorresponding IC₅₀ value for ACAT2. Most desirably, a selectiveinhibitor of ACAT1 has an IC₅₀ value for ACAT1 which is at least oneorder of magnitude or at least two orders of magnitude lower than theIC₅₀ value for ACAT2.

Selective inhibitors of ACAT1 activity have been described. For example,Ikenoya, et al. ((2007) Atherosclerosis 191:290-297) teach that K-604has an IC₅₀ value of 0.45 μmol/L for human ACAT1 and 102.85 μmol/L forhuman ACAT2. As such K-604 is 229-fold more selective for ACAT1 thanACAT2. In addition, diethyl pyrocarbonate has been shown to inhibitACAT1 with 4-fold greater activity (IC₅₀ =44 μM) compared to ACAT-2(IC₅₀=170 μM) (Cho, et al. (2003) Biochem. Biophys. Res. Comm.309:864-872). Ohshiro, et al. ((2007) J. Antibiotics 60:43-51) teachselective inhibition for ACAT1 over ACAT2 with beauveriolides I (0.6 μMvs. 20 μM) and III (0.9 μM vs. >20 μM). In addition, beauveriolideanalogues 258, 280, 274, 285, and 301 exhibit ACAT1-selective inhibitionwith pIC_(H) values in the range of 6 to 7 (Tomoda & Doi (2008) AccountsChem. Res. 41:32-39). Lada, et al. ((2004) J. Lipid Res. 45:378-386)teach a compound (designated therein as Compound 1A), and derivativesthereof (designated Compounds 1B, 1C, and 1D), which inhibit ACAT1 moreefficiently than ACAT2 with IC₅₀ values 66- to 187-fold lower for ACAT1than for ACAT2. Moreover, Lee, et al. ((2004) Bioorg. Med. Chem. Lett.14:3109-3112) teach methanol extracts of Saururus chinensis root thatcontain saucerneol B and manassantin B for inhibiting ACAT activity.Saucerneol B inhibited human ACAT-1 (hACAT1)and human ACAT-2 (hACAT2)with IC₅₀ values of 43.0 and 124.0 μM, respectively, whereas manassantinB inhibited hACAT-1 with an IC₅₀ value of 82.0 μM, only exhibiting 32%inhibition of hACAT2 at a very high concentration of 1 mM.

Desirably, ACAT1-selective inhibitors of the present invention have anIC₅₀ value in the range of 1 nM to 100 μM. More desirably,ACAT1-selective inhibitors of the invention have an IC₅₀ value less thanor equal to 100 μM. Most desirably, ACAT1-selective inhibitors of theinvention have an IC₅₀ value in the nM range (e.g., 1 to 999 nM).

In addition to the above-referenced ACAT1-selective inhibitors, it iscontemplated that any conventional drug screening assay can be employedfor identifying or selecting additional or more selective ACAT1inhibitors or derivatives or analogs of known ACAT1 inhibitors. See,e.g., Lada, et al. (2004) J. Lipid Res. 45:378-386. Such agents can beidentified and obtained from libraries of compounds containing pureagents or collections of agent mixtures. Examples of pure agentsinclude, but are not limited to, proteins, peptides, nucleic acids,oligonucleotides, carbohydrates, lipids, synthetic or semi-syntheticchemicals, and purified natural products. Examples of agent mixturesinclude, but are not limited to, extracts of prokaryotic or eukaryoticcells and tissues, as well as fermentation broths and cell or tissueculture supernates. In the case of agent mixtures, one may not onlyidentify those crude mixtures that possess the desired activity, butalso monitor purification of the active component from the mixture forcharacterization and development as a therapeutic drug. In particular,the mixture so identified may be sequentially fractionated by methodscommonly known to those skilled in the art which may include, but arenot limited to, precipitation, centrifugation, filtration,ultrafiltration, selective digestion, extraction, chromatography,electrophoresis or complex formation. Each resulting subfraction may beassayed for the desired activity using the original assay until a pure,biologically active agent is obtained.

Library screening can be performed in any format that allows rapidpreparation and processing of multiple reactions such as in, forexample, multi-well plates of the 96-well variety. Stock solutions ofthe agents as well as assay components are prepared manually and allsubsequent pipetting, diluting, mixing, washing, incubating, samplereadout and data collecting is done using commercially available roboticpipetting equipment, automated work stations, and analytical instrumentsfor detecting the signal generated by the assay. Examples of suchdetectors include, but are not limited to, luminometers,spectrophotomers, calorimeters, and fluorimeters, and devices thatmeasure the decay of radioisotopes. It is contemplated that any suitableACAT enzymatic assay can be used in such screening assays. Moreover,preclinical efficacy of ACAT1 inhibitors can be assessed usingconventional animal models of AD. Examples of conventional animal modelsof AD include but not be limited to models discussed in the scientificliterature and cited herein. Many of these models are models where micehave been genetically altered to either express certain genes or toablate expression of certain genes (transgenic mice). Such transgenicmouse models have been well-accepted for use in screening drugs forpotential therapeutic activity in humans and are commonly used in drugdevelopment.

As disclosed herein, there are a number of suitable molecules thatselectively inhibit the activity of ACAT1 without modulating theexpression of ACAT1. Accordingly, in one embodiment of the presentinvention, a “selective inhibitor of ACAT1” specifically excludesmolecules such as siRNA, antisense molecules, or ribozymes. However, inalternative embodiments, the ACAT1 selective inhibitor is a moleculewhich selectively inhibits the expression of ACAT1, without modulatingthe expression of ACAT2. While some RNAi molecules have been shown toinduce significant neurotoxicity in brain tissue (McBride, et al. (2008)Proc. Natl. Acad. Sci. USA 105:5868-5873), specific embodiments of thisinvention embrace one or more siRNA and/or microRNA molecules as theACAT1-selective inhibitor. As is conventional in the art, miRNA ormicroRNA refer to 19-25 nucleotide non-coding RNAs derived fromendogenous genes that act as post-transcriptional regulators of geneexpression. They are processed from longer (ca 70-80 nucleotide)hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer.MicroRNAs assemble in ribonucleoprotein complexes termed miRNPs andrecognize their target sites by antisense complementarity therebymediating down-regulation of their target genes. By way of illustration,target sequences for mouse ACAT1 microRNA molecules include, but are notlimited to, those listed in Table 2 as SEQ ID NOs:37-40. ArtificialmicroRNAs against human ACAT1 gene (e.g., GENBANK Accession No.NM_(—)000019, incorporated by reference) were also generated and shownto decrease human ACAT1 protein expression by 80% in human cells.Exemplary microRNA sequences targeting human ACAT1 include, but are notlimited, those listed in Table 4. In a similar manner, microRNA againstthe ACAT1 gene in primates (e.g., GENBANK Accession No. XM_(—)508738,incorporated by reference) can be developed, and used to selectivelyinhibit the expression of primate ACAT1.

SiRNA and/or microRNA molecules, which selectively inhibit theexpression of ACAT1, can be administered as naked molecules or viavectors (e.g., a plasmid or viral vector such as an adenoviral,lentiviral, retroviral, adeno-associated viral vector or the like)harboring nucleic acids encoding the siRNA and/or microRNA. Desirably, avector used in accordance with the invention provides all the necessarycontrol sequences to facilitate expression of the siRNA and/or microRNA.Such expression control sequences can include but are not limited topromoter sequences, enhancer sequences, etc. Such expression controlsequences, vectors and the like are well-known and routinely employed bythose skilled in the art. In particular embodiments, the siRNA and/ormicroRNA molecule is delivered by a non-viral delivery method, e.g.,liposome, nanoparticle, or liposome-siRNA-peptide complex (Pulford etal. 2010. PloS One 5:e11085).

The siRNA molecules of this invention may be modified by methods knownin the art to increase stability, increase resistance to nucleasedegradation or the like. These modifications are known in the art andinclude, but are not limited to modifying the backbone of theoligonucleotide, modifying the sugar moieties, or modifying the base. Inone embodiment, the invention features a siRNA molecule, wherein thesiRNA molecule includes a sense region and an antisense region andwherein the antisense region has a nucleotide sequence that iscomplementary to a nucleotide sequence or a portion thereof of RNAencoded by the ACAT1 gene and the sense region has a nucleotide sequencethat is complementary to the antisense region. In certain embodiments,the siRNA is composed of two nucleic acid molecules, e.g., a sense andantisense strand. In other embodiments, the siRNA is composed of onenucleic acid molecules, wherein the sense and antisense strand areconnected by a linker. In one embodiment, the purine nucleotides presentin the antisense region include 2’-deoxy-purine nucleotides. In anotherembodiment, the purine nucleotides present in the antisense regioninclude 2′-O-methyl purine nucleotides. In either of the aboveembodiments, the antisense region can include a phosphorothioateinternucleotide linkage at the 3′ end of the antisense region. In analternative embodiment, the antisense region includes a glycerylmodification at the 3′ end of the antisense region. In anotherembodiment of any of the above described siRNA molecules, anynucleotides present in a non-complementary region of the antisensestrand (e.g. overhang region) are 2′-deoxy nucleotides. See, e.g., U.S.Pat. No. 8,232,383; WO 00/44914; or WO 01/68836.

As indicated, selective inhibitors of ACAT1 find application in methodsfor decreasing the size and density of amyloid plaques, decreasingcognitive decline associated with amyloid pathology, and treating AD.Generally, such methods involve administering to a subject in need oftreatment a selective inhibitor of ACAT1 in an amount that effectivelyreduces the activity of ACAT1 by at least 60% to as much as 100%,including levels of inhibition between 60% and 100%. Subjects benefitingfrom treatment with an agent of the invention include subjects confirmedas having AD, subjects suspected of having AD, or subjects predisposedto have AD (e.g., subjects with a family history of Down's syndrome orones with a genetic predisposition to Alzheimer's disease). In thecontext of this invention, a subject can be any mammal including human,companion animals (e.g., dogs or cats), livestock (e.g., cows, sheep,pigs, or horses), or zoological animals (e.g., monkeys). In particularembodiments, the subject is a human.

While certain embodiments of this invention embrace in vivoapplications, in vitro use of agents of the invention are alsocontemplated for examining the effects of ACAT1 inhibition on particularcells, tissues or regions of the brain. In addition to treatment, agentsof the invention also find application in monitoring the phenotypicconsequences (e.g., rate of plaque formation or rate of cognitivedecline) of amyloid pathology in animal models of AD.

When used in therapeutic applications, an ACAT1-selective inhibitor ofthe invention will have the therapeutic benefit of decreasing the sizeand density of amyloid plaques in the subject, decreasing or slowing thecognitive decline associated with amyloid pathology in the subject,and/or treating AD in the subject as compared to subjects not receivingtreatment with the ACAT1-selective inhibitor. An ACAT1-selectiveinhibitor of the invention is expected to decrease the size and densityof amyloid plaques in a subject by any amount from 10% to 60% or more ascompared to an untreated subject. Similarly, an ACAT1-selectiveinhibitor of the invention is expected to decrease or slow the cognitivedecline associated by amyloid pathology by from any amount from 10% to60% or more as compared to an untreated subject (e.g., as determined bycommonly applied tests that would include but be limited to the BlessedInformation-Memory-Concentration Test, the BlessedOrientation-Memory-Concentration Test, and the Short Test of MentalStatus, or the Mini-Mental State Examination). Cognitive assessment caninclude monitoring of learning and retaining new information (e.g., doesthe subject have trouble remembering recent conversations, events,appointments; or frequently misplace objects), monitoring handling ofcomplex tasks (e.g., can the subject follow a complex train of thought,perform tasks that require many steps such as balancing a checkbook orcooking a meal), monitoring reasoning ability (e.g., is the subject ableto respond with a reasonable plan to problems at work or home, such asknowing what to do if the bathroom flooded), monitoring subject'sspatial ability and orientation (e.g., can the subject drive, organizeobjects around the house, or find his or her way around familiarplaces), and/or monitoring language (e.g., does the subject havedifficulty finding words to express what he or she wants to say and withfollowing conversations). Based upon a decrease in the observed and ormeasured signs and symptoms of AD, it is expected that AD will beprevented or slowed in a subject receiving treatment with an agent ofthe present invention, thereby treating the AD.

Successful clinical use of an ACAT1-selective inhibitor can bedetermined by the skilled practitioner, such as a clinician orveterinarian, based upon routine clinical practice, e.g., by monitoringcognitive decline via methods disclose herein, monitoring or measuringlevels of functional activities (e.g., the Functional ActivitiesQuestionnaire), and monitoring or measuring levels of sensory impairmentand physical disability according to methods known in the art.

For therapeutic use, ACAT1-selective inhibitors can be formulated with apharmaceutically acceptable carrier at an appropriate dose. Suchpharmaceutical compositions can be prepared by methods and containcarriers which are well-known in the art. A generally recognizedcompendium of such methods and ingredients is Remington: The Science andPractice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. LippincottWilliams & Wilkins: Philadelphia, Pa., 2000. A pharmaceuticallyacceptable carrier, composition or vehicle, such as a liquid or solidfiller, diluent, excipient, or solvent encapsulating material, isinvolved in carrying or transporting the agent in the subject from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be acceptable in the sense of being compatible withthe other ingredients of the formulation and must not be significantlyinjurious to the patient, although some level of toxicity can beexpected as well. One of skill in the art would understand how to ensurethat any agent used in a subject is one wherein the benefits to thesubject outweigh the risks to the subject. Given the serious nature ofAD, agents with some level of toxicity or risk to subject health couldbe tolerated and developed as a useful therapeutic agent to treat AD.

Examples of materials which can serve as pharmaceutically acceptablecarriers include sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose, and its derivatives,such as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatin; talc; excipients, such ascocoa butter and suppository waxes; oils, such as peanut oil, cottonseedoil, safflower oil, sesame oil, olive oil, corn oil and soybean oil;glycols, such as propylene glycol; polyols, such as glycerin, sorbitol,mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyllaurate; agar; buffering agents, such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters,polycarbonates and/or polyanhydrides; and other non-toxic compatiblesubstances employed in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Compositions of the present invention can be administered parenterally(for example, by intravenous, intraperitoneal, subcutaneous orintramuscular injection), topically, orally, intranasally,intravaginally, or rectally according to standard medical practices.

In certain embodiments of the present invention, the ACAT1-selectiveinhibitor is selectively delivered to the brain. For the purposes of thepresent invention, “selective delivery to the brain” or “selectivelydelivered to the brain” is intended to mean that the agent isadministered directly to the brain of the subject (e.g., by a shunt orcatheter; see, e.g., U.S. Patent Application No. 20080051691), to theperispinal space of the subject without direct intrathecal injection(see, e.g., U.S. Pat. No. 7,214,658), or in a form which facilitatesdelivery across the blood brain barrier thereby reducing potential sideeffects associated with ACAT1 inhibition in other organs or tissues. Inthis regard, formulation of the agent into a nanoparticle made bypolymerization of a monomer (e.g., a methylmethacrylate, polylacticacid, polylactic acid-polyglycolic acid-copolymer, orpolyglutaraldehyde) in the presence of a stabilizer allows passage ofthe blood brain barrier without affecting other organs with the agent.See, e.g., U.S. Pat. No. 7,402,573, incorporated herein by reference inits entirety. Moreover, an exemplary system for selectively deliveringmicroRNAs to the brain is the Adeno-Associated Virus (AAV) vectorsystem. See, e.g., Cearley & Wolfe (2007) J. Neurosc. 27(37):9928-9940.

The selected dosage level of an ACAT1-selective inhibitor will dependupon a variety of factors including the activity of the particular agentof the present invention employed, the route of administration, the timeof administration, the rate of excretion or metabolism of the particularagent being employed, the duration of the treatment, other drugs,compounds and/or materials used in combination with the particular agentemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and other factorswell-known in the medical arts.

A practitioner, such as a physician or veterinarian, having ordinaryskill in the art can readily determine and prescribe the effectiveamount of the pharmaceutical composition required based upon theadministration of similar compounds or after routine experimentaldetermination. For example, the physician or veterinarian could startdoses of an agent at levels lower than that required in order to achievethe desired therapeutic effect and gradually increase the dosage untilthe desired effect is achieved. This is considered to be within theskill of the artisan and one can review the existing literature on aspecific agent or similar agents to determine optimal dosing.

The invention is described in greater detail by the followingnon-limiting examples.

EXAMPLE 1 Methods

Mice. Mice were fed ad libitum with standard chow diet, maintained in apathogen-free environment in single-ventilated cages and kept on a 12hour light/dark schedule.

Generation of Acat1−/−Alz (A1−/Alz) and Acat2−/−/Alz (A2-−Alz) Mice.Acat1−/− and Acat2−/− mice (Meiner, et al. (1996) Proc. Natl. Acad. Sci.USA 93:14041-14; Buhman, et al. (2000) Nat. Med. 6:1341-1347) in C57BL/6background are known in the art. The 3XTg-Alz mice (Alzheimer's diseasemice) in hybrid 129/C57BL/6 background contain two mutant humantransgenes, hAPP harboring Swedish mutation (hAPPswe), and mutant htau(htau_(P301L)) under a neuron-specific promoter, and contain theknock-in mutant presenilin 1 (PS1_(M146V)) (Oddo, et al. (2003) Neuron39:409-421).

Mouse Tissue Isolation. Animals were sacrificed by CO₂ asphyxiation. Thebrains, adrenals and livers were rapidly isolated. Mice brains weredissected into various regions on ice within 5 minutes and were eitherused fresh (for ACAT enzyme activity assay) or were rapidly frozen ondry ice for other usage.

ACAT Activity Assay, Immunoprecipitation (IP) and Immunoblot Analyses.Freshly isolated tissue samples were homogenized on ice in 50 mM Tris, 1mM EDTA, pH 7.8 and solubilized in detergent using 2.5% CHAPS and 1 MKCl. The homogenates were centrifuged at 100,000 g for 45 minutes. Thesupernatants were used for ACAT activity assay in mixed micelles, andfor IP and immunoblot analyses (Chang, et al. (1998) J. Biol. Chem.273:35132-35141; Chang, et al. (2000) J. Biol. Chem. 275:28083-28092).

RNA Isolation, RT-PCR, and Real-Time PCR. Total RNA was isolated withTRIZOL reagent (Invitrogen), stored at −80° C., and used for RT-PCRexperiments, using the protocol supplied by the manufacturer. Real-timePCR was performed using the DYNAMO HS SYBR Green qPCR kit (New EnglandBiolabs). Relative quantification was determined by using the delta CTmethod (Pfaffl, et al. (2002) Nucleic Acids Res. 30:e36). Mouse ACAT1and human APP primers were designed using Oligo 4.0 Primer AnalysisSoftware. Mouse ACAT2, neurofilament 120-kD (NF120), GAPDH primerssequences are known in the art (Sakashita, et al. (2003) Lab. Invest.83:1569-1581; Kuwahara, et al. (2000) Biochem. Biophys. Res. Commun.268:763-766; Pan, et al. (2007) BMC Mol. Biol. 8:22). Sequences ofprimers used herein are listed in Table 1.

TABLE 1 Am- SEQ plicon ID Gene Size Sense/Antisense (5′->3′) NO: ACAT1274 AGCCCAGAAAAATTTCATGGACACATACAG  1 CCCTTGTTCTGGAGGTGCTCTCAGATCTTT  2ACAT2 530 TTTGCTCTATGCCTGCTTCA  3 CCATGAAGAGAAAGGTCCACA  4 GAPDH 186ATGGTGAAGGTCGGTGTG  5 CATTCTCGGCCTTGACTG  6 NF120 382 ACGGCGCTGAAGGAGATC 7 GTCCAGGGCCATCTTGAC  8 HUMAN 260 CCCACTGATGGTAATGCTGGC  9 APPGGAATCACAAAGTGGGGATGG 10 ABCA1  96 GGTTTGGAGATGGTTATACAATAG 11TTGTTTCCCGGAAACGCAAGTC 12 ABCG1  85 AGGTCTCAGCCTTCTAAAGTTCCTC 13TCTCTCGAAGTGAATGAAATTTATCG 14 ABCG4 541 CTGTCCTATTCCGTGCGGGA 15GGGACTTCATGAGGGACACCACTT 16 APOE 130 AGCCAATAGTGGAAGACATGCA 17GCAGGACAGGAGAAGGATACTCAT 18 CYP46A1 266 CAGTGAAGGTCATGCTGGAG 19CGCAATGAAGAAGGTGACAA 20 HMGR  69 TCTGGCAGTCAGTGGGAACTATT 21CCTCGTCCTTCGATCCAATTT 22 HMGS  77 GCCGTCAACTGGGTCGAA 23GCATATATAGCAATGTCTCCTGCA 24 HPRT  91 TTGCTCGAGATGTCATGAAGGA 25AGCAGGTCAGCAAAGAACTTATAGC 26 LDLR  68 CTGTGGGCTCCATAGGCTATCT 27GCGGTCCAGGGTCATCTTC 28 LRP  95 TGGGTCTCCCGAAATCTGTT 29ACCACCGCATTCTTGAAGGA 30 SREBP1 121 AACCAGAAGCTCAAGCAGGA 31TCATGCCCTCCATAGACACA 32 SREBP2 150 GTGGAGCAGTCTCAACGTCA 33TGGTAGGTCTCACCCAGGAG 34 SQS 137 CCAACTCAATGGGTCTGTTCCT 35TGGCTTAGCAAAGTCTTCCAACT 36

The PCR reaction conditions for amplification of ACAT1, ACAT2, GAPDH,NF120 and Human APP included an initial denaturation at 94° C. for 5minutes. Subsequently, 40 cycles of amplification were performed whichincluded: denaturation at 94° C. for 10 seconds, annealing at 56° C. for20 seconds, and elongation at 72° C. for 29 seconds. Amplificationconditions for the remaining primers listed in Table 1 were aspreviously described (Van Eck, et al. (2003) J. Biol. Chem.278:23699-23705).

In Situ Hybridization, Immunohistochemical and Thioflavin S Staining. Insitu hybridization was performed using standard procedures (Poirier, etal. (2008) J. Biol. Chem. 283:2363-2372) Immunohistochemistry wasperformed according to standard methods (Oddo, et al. (2003) supra).Thioflavin S staining was according to the protocol as described(Guntern, et al. (1992) Experientia 48:8-10), using free-floatingsections. Confocal analysis of thioflavin S-positive amyloid depositswas performed using known methods (Dickson & Vickers (2001) Neuroscience105:99-107).

Preparation of Brain Homogenates and Immunoblot Analysis of APP and ItsFragments, and Human Tau. Brain homogenates were prepared in sucrosebuffer with protease inhibitors at 4° C. according to a publishedprotocol (Schmidt, et al. (2005) Methods Mol. Biol. 299:267-278).Aliquots of homogenates were quickly frozen on dry ice and stored at−80° C. Upon usage, frozen homogenates were thawed on ice andcentrifuged for 1 hour at 100,000 g at 4° C.; the supernatants containedsoluble proteins including sAPPα and sAPPβ, while the pellet containedmembrane-associated, insoluble proteins including full-length APP, CTFα,CTFβ, etc. Immunoblot analysis of APP and its fragments was according to(Cheng, et al. (2007) J. Biol. Chem. 282:23818-23828). The followingantibodies were used: anti-human-Aβ 6E10 (1:5000) (Covance),anti-human-APP 369 antiserum (1:1000), anti-human-tau HT7 (1:1000)(Pierce), anti-human-tau phosphorylated at Ser202 AT8 (1:1000) (Peirce),monoclonal anti-HMG-CoA reductase IgG-A9 (1:3) (obtained from ATCC), andβ-actin (1:5000) (Sigma). Densitometric analysis was performed using NIHImage software.

Aβ Analysis by ELISA. Samples were prepared according to a standardprotocol (Schmidt, et al. (2005) Methods Mol. Biol. 299:279-297), thenloaded undiluted or diluted 5-10 fold onto the “human β amyloid (1-40)”or “human β Amyloid (1-42)” ELISA plate (Wako), and analyzed accordingto the protocol provided by the manufacturer (Wako).

Contextual Fear Conditioning. Contextual fear conditioning was performedaccording to a published protocol (Comery, et al. (2005) J. Neurosci.25:8898-8902; Jacobsen, et al. (2006) Proc. Natl. Acad. Sci. USA103:5161-5166). The auditory cue was from e2s (London, U.K.). GoldWavesoftware program was used to edit the auditory cue; Winamp software wasused to play the cue sound using the speakers. The digital sound levelmeter (RadioShack) was used to adjust the cue sound level to 87 dB. Eachmouse's behavior was recorded using a computer webcam (QuickCam fromLogitech) and ANY-maze recording software. The videos were analyzed forfreezing behavior, using time sampling at 5 second intervals.

Sterol Composition Analysis in Mice Brains. Mice forebrains werehomogenized and extracted using chloroform:methanol (2:1) (at 12 mlfinal vol. per mouse brain), dried down under nitrogen, and re-dissolvedin methanol. Ten percent of the sample was placed in a 2 ml GC/MSautosampler vial, dried down, and trimethyl-silyl derivatized overnightat room temperature with 0.5 ml TRI-SIL TBT (Pierce). One microliter ofderivatized sample (or 0.1 μl for cholesterol measurements) was injectedinto a Shimadzu QP 2010 GC-Mass instrument. GC/MS analysis of sterolswas performed according to known methods (Ebner, et al. (2006)Endocrinology 147:179-190) with modifications, using selected ionmonitoring (cholesterol: 24 329, 353, 368, 458; desmosterol: 441,lanosterol: 393; 24S-hydroxycholesterol: 413) and standard curves forquantification.

Sterol, Fatty Acid and Cholesterol Ester Synthesis in Mice Brains.Sterol and fatty acid synthesis in mice brains was measured according toknown methods (Reid, et al. (2008) J. Neurosci. Methods 168:15-25). Asimilar method was developed to measure cholesterol esterification from³H-cholesterol in vivo: mice were anesthetized with ketamine xylazine(0.1 ml/30 g body weight), and mounted onto a Kopf stereotaxicinstrument. After sagittal skin incision, ³H cholesterol at 10 μCi/mouseprepared in 3 μl of 5 mM methyl beta-cylodextrin in PBS was injectedinto the right lateral ventricle with a glass syringe (in 2 minutes).Mice were kept in cages for 3 hours, and then euthanized by CO₂ gas. Theforebrains were removed; lipids were extracted and redissolved inmethanol as described earlier. Ten percent of the redissolved sample wasanalyzed by TLC, using plates from Analtech, and solvent systemhexanes:ethyl ether (anhydrous): acetic acid (60:40:1). The cholesteroland 3H cholesterol ester (CE) bands were scraped off the TLC plate andcounted. Percent cholesterol esterification was determined by dividingthe CE count by the total ³H-cholesterol count.

Sterol Synthesis and Cholesterol Esterification in Primary Neuronal CellCulture. Hippocampal neurons were isolated from A1+/Alz and A1−/Alz miceat postnatal day 5 according to standard protocols (Brewer (1997) J.Neurosci. Methods 71:143-155; Price & Brewer (2001) In Protocols forNeural Cell Culture. Fedoroff & Richardson, editors. Totowa, N.J.:Humana Press, Inc. 255-264). Cells were seeded in 6-well dishes intriplicate at 300,000 cells/well, and grown in 3 ml/well Neurobasal Amedium with 1× B27, 0.5mM L-Gln and 5 ng/ml FGF for 14 days. ‘Half ofthe medium was replaced with fresh media once every 7 days. Forty-eighthours after the second media replacement, 50 μCi of [³H] sodium acetate(100 mCi/mmol) in phosphate-buffered saline (PBS) was added per well for3 hours. Lipids in cells and in media were extracted, saponified, andanalyzed by using the same TLC system described herein. To minimizesterol oxidation, samples were protected from light and heat duringlipid extraction, and were analyzed without storage. To improveseparation, after sample loading, the TLC plate was placed under vacuumfor 30 minutes prior to chromatography. ³H-labeled sterol bands wereidentified based on iodine staining of unlabeled sterols added tosamples prior to lipid extraction. Rf values: lanosterol, 0.5;cholesterol, 0.38; 24SOH: 0.2. The bands were, scraped off and counted.For each labeled sterol, the counts present in cells and in media wereadded to calculate the synthesis rate for that sterol. Cholesterolesterification in intact cells was conducted according to establishedmethods (Chang, et al. (1986) Biochemistry 25:1693-1699); the ³H-oleatepulse time was 3 hours.

Statistical Analysis. Statistical comparisons were made by using atwo-tailed, unpaired Student's-test. The difference between two sets ofvalues was considered significant when the P value was less than 0.05.Symbols used: *p<0.05; **p<0.01; ***p<0.001.

EXAMPLE 2 ACAT Expression in Mouse Brains

It has not been previously reported as to whether brain tissue has ACATenzyme activity. Therefore, to examine this, brain homogenates wereprepared from wild-type, Acat1−/−(A1−) and Acat2−/−(A2−) mice. Thisanalysis indicated that brain tissue of wild-type and A2− mice containedcomparable ACAT enzyme activity, while A1− brain tissue containednegligible activity. Various brain regions prepared from wild-type miceall contained ACAT activities, while regions examined in A1− mice braincontained no measurable activity. Mouse ACAT1 is a 46-kDa protein(Meiner, et al. (1997) J. Lipid Res. 38:1928-1933). Immunoblot analysisshowed that in homogenates prepared from mouse brain (but not from othermouse tissues), a non-ACAT1 protein band appeared in the 46-kDa region;the presence of this non-specific band precluded the use ofimmunoblotting or histochemical staining to identify ACAT1 in the mousebrain. To unambiguously identify ACAT1 protein, immunoprecipitation (IP)experiments were performed using detergent solubilized wild-type mousebrain extracts. The results of the IP experiment showed that ACATactivity could be efficiently immunodepleted by ACAT1-specificantibodies, but not by control antibodies. Immunoblot analysis of theimmunoprecipates was then performed. The results showed that inhomogenates from wild-type mouse brain regions, the ACAT1 antibodiesspecifically identified a 46-kDa-protein band; control experimentsshowed that this band was absent in homogenates prepared from theadrenals and brains of A1− mice. This result indicated that ACAT1 isexpressed in mouse brain tissue and is the major ACAT isoenzyme.

To determine the distribution pattern of ACAT1 mRNA in mouse brain, insitu hybridization experiments were performed. Both hippocampus andcortex contained ACAT1 mRNA; with hippocampus showing a stronger signal.Other ACAT1 positive regions included choroid plexus, medial habenularnucleus, amygdala, and rostral extension of the olfactory peduncle.Subsequently, hippocampus-rich regions and cortex-rich regions wereisolated from wild-type mice, and ACAT1 mRNA levels were compared byreal-time PCR. The result validated the in situ hybridizationexperiment, and showed that ACAT1 mRNA was -2-fold higher in hippocampusthan in cortex. A separate, RT-PCR experiment using ACAT2-specificprimers showed that only the thalamus-rich region, but no other brainregion, expressed low but detectable ACAT2 mRNA. It has similarly beenshown that monkey brain tissue exhibits nearly undetectable levels ofACAT2 mRNA (Anderson, et al. (1998) J. Biol. Chem. 273:26747-26754).

EXAMPLE 3 ACAT1-Deficient Alzheimer's Mice (A1−/Alz Mice)

While non-selective ACAT inhibition has suggested a role for ACATactivity in AD pathology, it had not been shown whether the effects ofthe ACAT inhibitor are due to activity to inhibit ACAT activity alone,or due to activity on other biological process(es) in mouse braintissue, or due to a combination of both. Accordingly, a genetic approachwas employed to definitively assess the role of each ACAT isoenyzme inthe pathology of AD.

To carry out this analysis, a triple transgenic AD mouse model(3XTg-Alz; Oddo, et al. (2003) supra), which has been shown to be aneffective research tool for studying AD (Morrissette, et al. (2009) J.Biol. Chem. 284:6033-6037) was crossed to an ACAT1 (A1−) or ACAT2 (A2−)knock-out mouse (Buhman, et al. (2000) Biochim. Biophys. Acta1529:142-154) and amyloid pathology development was monitored in themice with or without ACAT. The results showed that, at 4 months of age,when compared to the control mice, the intraneuronal amyloid-β load inthe hypocampal neurons was significantly decreased in the A1−/Alz mice,but not in the A2−/Alz mice. At 17 months of age, when compared to thecontrol mice, the sizes and densities of the amyloid plaques weresignificantly decreased in the A1/Alz− mice. Behavioral analysis showedthat ACAT1 deficiency rescued the cognitive decline manifested in themouse model of AD. These results showed that ACAT1 gene inactivationcaused a significant decrease in amyloid pathology in a mouse model forAD. Thus, ACAT1, but not ACAT2, is a therapeutic target for treating AD.

EXAMPLE 4 Effect of A1− on Aβ Deposition/hAPPswe Processing, and on hTau

To investigate the effect of inactivating ACAT1 on amyloid and taupathologies in the 3XTg-Alz mice, A1−/Alz mice were examined used thehuman specific anti-Aβ antibody 6E10 to perform intraneuronalimmunostaining in the CA1 region of hippocampal brain tissue of4-month-old mice. Results showed that the staining was significantlydiminished (by ˜78%) in the A1−/Alz mice. An enzyme-linked immunosorbentassay (ELISA) was next used to measure the total Aβ40 and Aβ42 levels inmouse brain homogenates at 17 months of age. Results showed that the Aβ42 levels were significantly decreased (by ˜78%) in A1−/Alz mice; theAβ40 levels were also decreased, but the difference observed was notstatistically significant. Control experiments showed that the brains ofnontransgenic mice did not contain measurable Aβ. Thioflavin S wassubsequently used to stain amyloid plaques in Alz mouse brains at 17months of age. The results showed that in A1−/Alz mice the amyloidplaque load in the hippocampal region of brain tissue was significantlyreduced (by ˜77%); in the cortex region, the amyloid plaque load inthese mice showed a trend toward decreasing (p=0.17).

The effect of A1− on human APP processing in 4-month-old Alz mice wasalso analyzed. The human-specific anti-Aβ antibody 6E10 was used todetect full-length human APPswe (hAPP), and its proteolytic fragmentssAPPα (hsAPPα) (soluble APP fragment produced by a secretase cleavage)and CTFβ (hCTFβ) (C-terminal APP fragment produced by β secretasecleavage) (Thinakaran & Koo (2008) supra). The results showed that inA1−/Alz mice, hsAPPα and hCTFβ levels were decreased (by ˜67% and by˜37%, respectively). Unexpectedly, the hAPP level was also significantlyreduced (by ˜62%). In contrast to the hAPP protein levels, there was nodifference in hAPP mRNA levels between the A1+/Alz mice and the A1−/Alzmice. hAPP is synthesized in the endoplasmic reticulum in its immatureform (with a molecular weight of ˜105-kDa); the immature form moves fromthe endoplasmic reticulum to the Golgi via a secretory pathway (Cai, etal. (2003) J. Biol. Chem. 278:3446-3454), and becomes highlyglycosylated (mature form has a molecular weight of ˜115-kDa)(Weidemann, et al. (1989) Cell 57:115-126; Oltersdorf, et al. (1990) J.Biol. Chem. 265:4492-4497; Thinakaran, et al. (1996) J. Biol. Chem.271:9390-9397). Thus, the effects of A1− on the immature and the matureforms of hAPP in young Alz mice (of 25-day old) were examined. Theresults showed that A1− decreased both forms to approximately the sameextent (by ˜52-54%), indicating that the effect(s) of A1− occur beforenewly synthesized hAPP exits the endoplasmic reticulum.

The Alz mice express both hAPP and endogenous (mouse) APP. It ispossible that A1− may affect both the hAPP and the mAPP levels. Toinvestigate the total APP levels in Alz mice, a different antibody(antiserum 369) was used, which recognizes the C-terminal fragments ofboth hAPP and mAPP (Buxbaum, et al. (1990) Proc. Natl. Acad. Sci. USA87:6003-6006). The results showed that there was no detectabledifference in the total APP levels between the non-Tg, the A1+/Alz, andthe A1−/Alz mice, indicating that in the Alz mice strain, the hAPP isnot over-expressed, when compared to the endogenous mAPP protein level.mAPP processing was also examined in mice that did not contain the hAPPgene. In these mice, A1− also did not affect the levels of mAPP (and itshomolog APLP2 (Slunt, et al. (1994) J. Biol. Chem. 269:2637-2644)), orany of the proteolytic fragments derived from mAPP. These results led tothe conclusion that A1− only reduced the hAPP level, and not the mAPPlevel. It is known that subtle sequence differences exist between hAPPand mAPP, and these differences may play an important role in causingdifferential fates of hAPP and mAPP (Du, et al. (2007) J. Pharmacol.Exp. Ther. 320:1144-1152; Muhammad, et al. (2008) Proc. Natl. Acad. Sci.USA 105:7327-7332). The results herein are in contrast to previousreports that indicated that an ACAT inhibitor affected the proteolyticprocessing of mouse APP, in addition to affecting the processing of hAPP(Hutter-Paier et al. (2004) supra). The discrepancy between the resultsherein and those of Hutter-Paier, et al. may be attributable tooff-target or side effect(s) of the ACAT inhibitor used in their study.

Tau pathology is one of the hallmarks of AD. Accordingly, the effect ofA1− on mutant human tau (htau) was analyzed in 3XTg-Alz mice. Theresults showed that at 4 months of age, A1− mice exhibited a significantdecrease in htau (by ˜57%), but at 17 months of age, A1− mice had anincreased level of hyperphosphorylated htau. No significant change wasobserved in the number of hippocampal neurofibrillary tangles betweenthe A1+/Alz and the A1−/Alz mice. These results indicated that A1− doesnot attenuate tau pathology in Alz mice.

EXAMPLE 5 Effect of A1− on Cognitive Deficits of Alz Mice

To examine effects of agents on cognitive deficits in Alz mice,contextual (hippocampus dependent) and cued (amygdala dependent) memorytests were performed on age-matched (2, 9 and 12 months old) A1+/Alz,A1−/Alz and Non-Tg mice. The results showed that mice of all threegenotypes at different ages were able to learn equally well. Incontextual memory testing, there was no difference among these mice at 2months of age. However, at 9 and 12 months, when compared to Non-Tgmice, the A1+/Alz mice exhibited a ˜50% deficit, while the A1−/Alz miceexhibited no deficit. In cued memory tests, there was again nodifference among the mice at 2 months. Yet, at 9 months, when comparedto Non-Tg mice, the A1+/Alz mice exhibited a trend toward a decline;however, the difference was not statistically significant. At 12 months,a statistically significant memory decline in the A1+/Alz mice wasobserved. In contrast, the A1−/Alz mice exhibited no deficit at either 9or 12 months age. These results indicate that A1− ameliorated thehippocampal- and amygdala-dependent cognitive deficits in Alz mice at9-12 months of age. As a control, contextual and cued tests were alsoperformed on A2+ and A1− mice in the C57BL/6 background at 9 and 12months of age. The results showed that the A1+ and the A1− mice wereable to learn equally well in either contextual or cued memory tests,wherein the difference between the A1− mice and the A1+ mice was notstatistically significant.

EXAMPLE 6 Effects of A1− on Sterol Metabolism in Alz Mouse Brains

ACAT is an important enzyme in cellular cholesterol homeostasis. It wascontemplated that A1− may decrease hAPP content by affecting sterolmetabolism in Alz mice brains. To demonstrate this, sterol fractionsfrom A1+/Alz and A1−/Alz mouse brains were isolated and analyzed byGC/MS. The results showed that at 4 months of age, ACAT1 deficiencycaused a ˜13% decrease in cholesterol content (p=0.04) and a ˜32%increase in 24SOH content (p=0.007), without causing significant changesin either lanosterol or desmosterol content. A similar decrease incholesterol content of the A1−/Alz mouse brain was observed when acolorimetric enzyme assay kit (Wako) as used to determine freecholesterol. It was also found that in the brains of 2-month-old Alzmice, A1 deficiency caused a -10% decrease in cholesterol content and a˜23% increase in 24SOH content. Subsequently, the relative sterolsynthesis and fatty acid synthesis rates were compared in the brains ofthese mice in vivo. The results showed that A1− caused a ˜28% decreasein the sterol synthesis rate (p=0.04) without significantly changing thefatty acid synthesis rate. In mouse brain, cholesteryl ester contentsare reported to be very low (Yusuf & Mozaffar (1979) J. Neurochem.32:273-275; Liu, et al. (2009) Proc. Natl. Acad. Sci. USA106:2377-2382). An attempt was made to measure CE in A1+ mouse braintissue by separating the CE fraction from the free cholesterol fractionusing column chromatography and determining the cholesterol content inCE by GC/MS after CE was saponified. While the low level of CE preventeda reliable measurement, the results suggested that CE might be presentat no more than 1% of the total cholesterol mass in mouse brain tissue.Using a similar procedure to determine the 24SOH ester content, it wasestimated that no more than 1% of total 24S0H was esterified in thebrain. These results are consistent with the finding that ACAT prefersto use cholesterol, as opposed to various oxysterols, as its enzymaticsubstrate (Zhang, et al. (2003) J. Biol. Chem. 278:11642-11647; Liu, etal. (2005) Biochem. J. 391:389-397).

To demonstrate the functionality of ACAT1 in the intact mouse brain, aprocedure was developed to measure CE synthesis in vivo by injecting³H-labeled cholesterol (as a cyclodextrin complex) into intact mousebrain. The ³H-CE produced in A1+ and A1− mice was monitored 3 hoursafter injection. The results of this experiment showed that in A1+/Alzmice, a small percentage of ³H-cholesterol was converted to ³H-CE (0.56%in 3 hours); in contrast, such conversion was not detectable in theA1−/Alz mouse brain. This result demonstrated that ACAT1 in intact mousebrain can synthesize CE, although at a low rate.

The data herein indicated that in Alz mouse brain, A1− leads to anincreased 24SOH level, which in turn leads to a down-regulation of thesterol synthesis rate. Studies in cell culture have suggested that 24S0Hmay down-regulate sterol synthesis by two mechanisms, namely by blockingtranscriptional activations of SREBP2 target genes, and/or increasingthe degradation rate of HMGR protein (Goldstein, et al. (2006) Cell124:35-46). To test the first possibility, the mRNA levels of variousSREBP2 and LXR target genes (i.e., HMGR, HMGS, SQS, LRP, LDLR, SREBP2,SREBP1, APOE, ABCA1, ABCG1, ABCG4, and CYP46A1) were compared in theA1+/Alz and the A1−/Alz mouse brain. This analysis indicated nosignificant alterations in the expression levels of these genes in thebrains of mice with or without ACAT1. To test the second possibility,immunoblot analysis was performed on brain homogenates prepared from theAlz mice with or without ACAT1. The results showed that HMGR proteincontent was decreased by ˜65% in A1−/Alz mouse brain (p=0.0009), whilethe HMGR mRNA in A1− mouse brain was not changed. Additional resultsshowed that in Alz mice at 25-days of age, A1− caused a ˜62% decrease inHMGR protein content, demonstrating that the effect of A1− on HMGRcontent occurs in mice at a young age.

EXAMPLE 7 Biosynthesis of 24SOH in Hippocampal Neuronal Cell Cultures

The results described herein show that A1−/Alz mouse brain exhibitselevated 24SOH levels, indicating that in mouse neurons, A1− may causean increase in the biosynthesis of 24SOH. In so far as cultured neuronsisolated from brains have been shown to synthesize and secrete 24SOH(Russell, et al. (2009) supra; Kim, et al. (2007) J. Biol. Chem.282:2851-2861), a hippocampal neuronal cell culture system wasestablished from A1+/Alz and A1−/Alz mice to determine whether thesecells exhibit an increase in the biosynthesis of 24SOH. CE biosynthesiswas monitored in these neurons by incubation with labeled ³H-oleic acid.Upon entering cells, ³H-oleic acid is rapidly converted to ³H-CE byACAT. Both the A1+ cells and the A1− cells synthesize CE; however, A1−cells synthesize ³H-CE at a much reduced capacity compared to A1+ cells.The effect of A1− on 24SOH biosynthesis was subsequently analyzed byfeeding neurons with the sterol precursor ³H-actetate for 3 hours, thenisolating and analyzing the labeled sterols present in the cells andmedia. The results showed that A1− cells exhibited a reduced trend incholesterol synthesis rate; the difference observed between A1+]cellsand A1− cells approached but did not reach statistical significance(p=0.05). The 24SOH synthesis rate in A1− cells was significantlyincreased (by ˜27%). The ³H-sterols in the media of A1+ and A1− cellswas also examined. The results showed that the ³H-cholesterol contentwas not significantly different; in contrast, the ³H-24SOH content inA1− cells was significantly (˜56%) higher than that in A1+ cells. Thepercent of total ³H-sterols secreted into the media was calculated andit was found that neurons secreted only about 2% of total³H-cholesterol, but secreted 13-15% of total ³H-24SOH into the media.

The results herein demonstrate that A1− causes an increased 24SOHbiosynthesis rate in neurons. Mouse neurons maintained in cultureexpress CYP46A1 as a single 53-kDa-protein, which can be identified byimmunoblot analysis (Russell, et al. (2009) supra). It is possible thatthe increased synthesis of 24SOH observed in A1− neurons may be due toan increase in CYP46A1 protein content in these neurons. To determinethis, CYP46A1 protein content in A1+ and A1− neurons was analyzed byimmunoblot analysis. The results showed that the intensities of the53-kDa-protein band were comparable between the A1+/Alz and A1−/Alz celltypes. This result indicates that in hippocampal neurons, themechanism(s) involved in the A1− associated increase in 24SOH synthesisdoes not require an increase in CYP46A1 protein content.

EXAMPLE 8 24SOH Treatment of Alz Mouse Neurons Decreases hAPP ProteinContent

The observations made in intact A1−/Alz mouse brains (i.e., an increasein 24SOH content and a decrease in hAPP content) indicated that 24SOHmay decrease hAPP content in neurons. To test this, hippocampal neuronsfrom A1+/Alz mice were treated with 24SOH, and the hAPP protein contentand the HMGR protein content were monitored in parallel. It was foundthat 1 μM 24SOH rapidly decreased the protein content of both hAPP andHMGR (within 3 hours). A separate experiment showed that 1-5 μM 24SOHcaused a rapid decline in hAPP protein content without affecting itsmRNA level. This result indicates that accumulation of 24SOH in neuronsmay down-regulate hAPP protein content in vivo.

Thus, the current findings link cellular cholesterol trafficking withACAT1, CYP46A1, 24SOH synthesis, and HMGR at the endoplasmic reticulum.In neurons, cholesterol trafficking in and out of the endoplasmicreticulum occurs. The unnecessary buildup of unesterified cholesterol atthe endoplasmic reticulum (and other membranes) is toxic (Tabas (2002)J. Clin. Invest. 110:905-911; Warner, et al. (1995) J. Biol. Chem.270:5772-5778). To minimize cholesterol accumulation, ACAT1, a residentenzyme located at the endoplasmic reticulum (Chang, et al. (2006) Annu.Rev. Cell Dev. Biol. 22:129-157), removes a portion of endoplasmicreticulum cholesterol by converting it to CE. ACAT1 deficiency leads toan increase in the endoplasmic reticulum cholesterol pool and raises thesubstrate level for CYP46A1, another endoplasmic reticulum residentenzyme (Russell, et al. (2009) supra). This leads to an increase in24SOH biosynthesis in neurons. The increased 24SOH concentration leadsto rapid down-regulation of hAPP protein content, limiting its capacityto produce Aβ. 24SOH secreted by neurons can enter astrocytes and othercell types and lead to efficient down-regulation of HMGR and cholesterolbiosynthesis in these cells. Therefore, the beneficial effects of ACAT1inhibition on cholesterol biosynthesis and on amyloid pathology isattributed to its ability to increase 24SOH level in Alz mouse brains.Therefore, agents that inhibit ACAT1 enzyme activity or decrease ACAT1gene expression can ameliorate amyloid pathology, and have therapeuticvalue for treating AD in humans. These results also indicate that agentsthat increase the concentration of 24SOH may help to combat AD bydecreasing APP content in the brain. Such agent include, but are notlimited to, 24SOH itself.

EXAMPLE 9 MicroRNA-Mediated Inhibition of ACAT1 Expression

Artificial microRNA molecules were designed to target the 5′ end of thecoding sequence of mouse ACAT1 sequences listed in Table 2.

TABLE 2 SEQ microRNA ACAT1 Target Sequence ID NO: #52GGAGCTGAAGCCACTATTTAT 37 #53 CTGTTTGAAGTGGACCACATCA 38 #54CCCGGTTCATTCTGATACTGGA 39 #55 AACTACCCAAGGACTCCTACTGTA 40

For example, the pre-microRNAs (including sense, antisense and loopregions) of microRNAs #54 and #55 were 5′-TGC TGT CCA GTA TCA GAA TGAACC GGG TTT TGG CCA CTG ACT GAC CCG GTT CAC TGA TAC TGG A-3′ (SEQ IDNO:41) and 5′-TGC TGT ACA GTA GGA GTC CTT GGG TAG TTT TGG CCA CTG ACTGAC TAC CCA AGC TCC TAC TGT A-3′ (SEQ ID NO:42), respectively.

NIH-3T3 mouse fibroblasts were transiently transfected with one ofseveral rAAV vectors encoding EmGFP and microRNA (miR) #52, #53, #54 or#55. Forty-eight hours post-transfection, GFP-positive cells wereharvested by FACS. GFP-positive cells were washed then lysed in 10% SDSand syringe homogenized. Twenty microgram of protein per sample wassubjected to SDS-PAGE. After western blot analysis, bands werequantified with ImageJ. ACAT1 intensity was normalized to GAPDH as aloading control and expressed as relative intensity. The results of thisanalysis are presented in Table 3.

TABLE 3 Treatment Relative Intensity Mock Transfected 1.00 miR NegativeControl 1.02 miR #52 0.77 miR #53 0.56 miR #54 0.54 miR #55 0.39

This analysis indicated that microRNA molecules directed to mouse ACAT1sequences could effectively decrease mouse ACAT1 gene expression by morethan 50% compared to untreated controls.

Similarly, treatment of human HeLa cells or MCF-7 cells with either ofthe microRNAs listed in Table 4 (10 nM concentration for two days)decreased human ACAT1 protein expression by 80%.

TABLE 4 SEQ MicroRNA Sequence(5′->3′) ID NO: CAUGAUCUUCCAGAUUGGAGUUCUA43 UAGAACUCCAAUCUGGAAGAUCAUG 44

EXAMPLE 10 Clinical Assessment of Therapeutic Efficacy

A cohort of subjects fulfilling NINCDS-ADRDA criteria (McKhann, et al.(1984) Neurology 34:939-44) for probable or possible AD will berecruited. The median age of the sample group will be determined.Clinical diagnosis will be made independently by, e.g., a psychiatristand neurologist based on a checklist for symptoms of the disease withstrict adherence to NINCDS-ADRDA criteria. Cognitive assessment will berecorded by trained clinical research nurses using the MMSE (Mini MentalState Examination; Folstein et al. (1975) J. Psychiatric Res.12:189-98). Assessment will be followed a standardized protocol tomaximize inter-rater reliability. All subjects will be followed up atyearly intervals, for a period of up to three years or more with repeatMMSE on each occasion.

During the trial period, subjects will either receive regular doses ofan ACAT1-selective inhibitor or placebo. The rate of cognitive declinewill be based on the average slope of MMSE points change per year.Differences in the average annual MMSE decline in the whole group by thepresence or absence of the K variant of the ACAT1-selective inhibitorwill be assessed by the Mann-Whitney U test. The subjects will then begrouped into four categories depending on their baseline MMSE scores(e.g., >24; ≦24 and >16; ≦16 and >8; ≦8 points). Differences in theaverage annual MMSE decline in the four categories by the presence orabsence of the K variant of ACAT1-selective inhibitor will be initiallyassessed by independent t-tests. Linear regression analysis with theaverage annual MMSE decline as the dependent variable will then be usedto assess for confounding and effect modification by the independentvariables, e.g., MMSE at baseline, age, age of onset, and sex. It isexpected that the results of this analysis will indicate that subjectsreceiving the ACAT1-selective inhibitor will exhibit a decrease in therate or severity of cognitive decline as compared to subjects receivingplacebo.

EXAMPLE 11 Specificity of ACAT1 Inhibitors

It has been shown that when the ACAT inhibitor CP113818 or CI 1011 areadministered to AD mice, amyloid plaques are significantly reduced andcognitive deficits are rescued, suggesting that inhibiting ACAT mayprevent and/or slow down the progression of AD (Hutter-Paier, et al.(2004) supra; Huttunen & Kovacs (2008) supra; Huttunen, et al. (2009)supra). However, close comparison of the instant data and data of theprior art indicates that several important differences exist between theeffects of the ACAT inhibitors and the effects of ACAT1 gene expression.CP113818 inhibits the processing of both human APP and mouse APP,whereas CI 1011 decreases the mature/immature ratio of hAPP. Incontrast, A1− only caused a decrease in the full-length human APPprotein content and did not affect the mouse APP at any level or alterthe mature/immature ratio of hAPP. Another important difference is thatunlike the effect of A1−, CP113818 causes a reduction in the full-lengthhAPP content (Hutter-Paier, et al. (2004) supra). The differences inresults indicate that the ACAT inhibitors used in the prior art are notselective for ACAT1, as evidenced by the differences in results seenwith complete ablation of ACAT1 (AT1−).

ACAT is a member of the membrane bound O-acyltransferase (MBOAT) enzymefamily (Hofmann (2000) Trends Biochem. Sci. 25:111-112), which includessixteen enzymes with similar substrate specificity and similar catalyticmechanisms, but with diverse biological functions. In addition, manyACAT inhibitors are hydrophobic, membrane active molecules (Homan &Hamelehie (2001) J. Pharm. Sci. 90:1859-1867). When administrated tocells, it is likely that they partition into membranes at highconcentration, thereby perturbing membrane properties nonspecifically.Although CP113818 and CI 1011 are designated as ACAT inhibitors, theyalso may inhibit other enzymes in the MBOAT family, and/or interferewith other biological processes.

The present data shows that inactivating the ACAT1 gene alone issufficient to ameliorate amyloid pathology in the 3XTg-AD mouse model.In this mouse model, A1− acts to reduce Aβ load mainly by reducing thehAPP protein content. In this context, the action of A1− is similar tothat of cerebrolysin, a peptide mixture with neurotrophic effects. Ithas been shown that cerebrolysin reduces Aβ in an AD mouse model, mainlyby decreasing the hAPP protein content (Rockenstein, et al. (2006) J.Neurosci. Res. 83:1252-1261; Rockenstein, et al. (2007) ActaNeuropathol. 113:265-275). To further demonstrate that A1− leads to hAPPcontent reduction, it was shown that the brains of A1−/Alz mice containa significantly greater amount of 24SOH. Moreover, in neuron-richcultures, it was shown that 24SOH, when added to the medium, leads torapid decrease in hAPP protein content. It is possible that APP may actas a sterol sensing protein (Beel, et al. (2008) Biochemistry47:9428-9446); sequence analysis shows that APP contains three CRACmotifs, a consensus motif known to bind cholesterol (Epand (2008)Biochim. Biophys. Acta 1778:1576-1582). It is also possible thatcholesterol and/or oxysterol may directly interact with the hAPP proteinto accelerate its rate of degradation. Alternatively, 24SOH may actindirectly by reducing membrane cholesterol content.

The data presented herein also show that in mouse brain, A1− caused adecrease in HMGR protein and a decrease in cholesterol biosynthesis.This finding is consistent with previous analysis showing thatinhibition of ACAT in macrophages, or in CHO cells, increases the ER“regulatory sterol pool” that causes down-regulation of HMGR levels andSREBP processing (Tabas, et al. (1986) J. Biol. Chem. 261:3147-3155;Scheek, et al. (1997) Proc. Natl. Acad. Sci. USA 94:11179-11183).Studies have suggested that the “regulatory sterol” could be cholesterolitself, and/or an oxysterol derived from cholesterol; however, whetheroxysterol(s) plays important roles in regulating sterol biosynthesis inthe brain in vivo has been debated (Bjorkhem (2009) J. Lipid Res.50:S213-218).

To address this issue, it has been shown that knocking out the24-hydroxylase gene Cyp46a1 causes a near elimination in the 24SOHcontent, a decrease in cholesterol biosynthesis rate in the brain, and adecrease in cholesterol turnover in the brain; the total braincholesterol content in the Cyp46a1^(−/−) mice remained unchanged;Cyp46a1^(−/−) did not affect the amyloid pathology in an AD mouse model(Lund, et al. (2003) J. Biol. Chem. 278:22980-22988; Kotti, et al.(2006) Proc. Natl. Acad. Sci. USA 103:3869-3874; Halford & Russell(2009) Proc. Natl. Acad. Sci. USA 106:3502-3506). In contrast, use of acell-type non-specific promoter to drive the ectopic expression ofCyp46a1 in mouse brain shows that over-expressing Cyp46a1 causes atwo-fold increase in 24SOH content and significantly ameliorates amyloidpathology in the AD mice (Hudry, et al. (2010) Mol. Ther. 18:44-53). Inthis study, a reduction in the hAPP protein content was not observed;instead, a decrease in hAPP processing, an increase in SREBP2 mRNA, andno change in brain cholesterol content was demonstrated. The presentresults show that in the A1−/Alz mice, a 30% increase in 24SOH in braincholesterol content, a modest reduction in cholesterol biosynthesisrate, and a significant reduction in amyloid pathology occurred. TheCyp46a1 gene knockout or Cyp46a1 overexpression in mice may haveproduced compensatory effects that did not occur in the A1− mice, andvice versa; thus a direct comparison of the results described above isdifficult. On the other hand, the combined results suggest that 24SOHmay play an auxiliary, but not an obligatory, role in affectingcholesterol metabolism and amyloid biology, and its effects may becell-type dependent. Based on other evidence, it has been independentlyproposed that a given oxysterol may play auxiliary but not obligatoryroles in regulating cellular cholesterol homeostasis (Brown & Jessup(2009) Mol. Aspects Med. 30:111-122).

The instant data demonstrate a link between ACAT1, CYP46A1, 24SOHsynthesis, and HMGR at the level of the endoplasmic reticulum (ER) incellular cholesterol trafficking. The unnecessary buildup ofunesterified cholesterol at the ER (and other membranes) is toxic(Warner, et al. (1995) J. Biol. Chem. 270:5772-5778; Tabas (2002) J.Clin. Invest. 110:905-911). In order to minimize cholesterolaccumulation, ACAT1, a resident enzyme located at the ER (Sun, et al.(2003) J. Biol. Chem. 278:27688-27694), removes a portion of ERcholesterol by converting it to CE. A1− leads to an increase in the ERcholesterol pool and raises the substrate level for CYP46A1, another ERresident enzyme. This leads to an increase in 24SOH biosynthesis inneurons. The increased 24SOH and/or cholesterol concentration in the ERleads to rapid down-regulation of hAPP protein content, thereby limitingits capacity to produce Aβ. 24SOH secreted by neurons can enterastrocytes and other cell types, and lead to efficient down-regulationof HMGR and cholesterol biosynthesis in these cells. Therefore, thebeneficial effects of A1− on cholesterol biosynthesis and on amyloidpathology in AD mouse brains is attributed to an increase(s) in ERcholesterol and/or 24SOH level in the neurons. Barring the possible sideeffects caused by altering cholesterol metabolism in the brain, theinstant data indicate that agents that selectively and specificallyinhibit ACAT1 enzyme activity or decrease ACAT1 gene expression canameliorate amyloid pathology, and have therapeutic value for treating ADin humans.

EXAMPLE 12 Effect of Recombinant Adeno-Associated Virus Expressing Acat1siRNA

Four different siRNA sequences (#52-#55; Table 2) targeting the mouseAcat1 gene were inserted into an endogenous mouse microRNA (miR)scaffold using Invitrogen's RNAi design tool. The artificial miRs wereligated into the mammalian expression vector pcDNA6.2-GW/EmGFP-miR.These Acat1miR constructs were tested along with a negative control (NC)miR (5′-TACTGCGCGTGGAGACG-3′; SEQ ID NO:9), which does not match thesequence of any known vertebrate gene, in NIH-3T3 mouse fibroblasts. ThemiRs were delivered to the cells using a standard cDNA transfectionprotocol. The results showed that two of the Acat1 miRs (containing thesiRNA sequence #54, 5′-TACAGTAGGAGTCCTTGGGTA-3′; SEQ ID NO:10, andsequence #55, 5′-TCCAGTATCAGAATGAACCGGG⁻3′; SEQ ID NO:11) were effectivein causing 50-60% reduction in the ACAT1 protein content in treatedmouse 3T3 fibroblasts.

These two Acat1 miR sequences and the NC miR sequence were alsosubcloned into a rAAV backbone vector (AAV-6P-SEWB) that contained theneuron-specific hSyn promoter (Sibley, et al. (2012) Nucl. Acids Res.Doi:10.1093/nar/gks712). This vector contained a strong and cell-typenonspecific promoter that expresses Acat1 miRs in any cell type wherethe viral genome is expressed. For identification purpose, it alsoco-expresses the GFP with the miRs. These three constructs were used toproduce three recombinant AAV viruses. To test the efficacy andspecificity of these viruses, cultured primary hippocampal neuronsisolated from the triple transgenic Alzheimer neurons from AD mice(AD/Acat1+/+ mice) were treated with the NC AAV, or with AAV thatexpressed miR containing siRNA Acat1 #55. Two weeks after viralinfection, the effects of AAVs on cholesteryl ester biosynthesis weretested in neurons. The results showed that the AAV harboring siRNA Acat1#55 reduced cholesteryl ester biosynthesis by more than 50% (P<0.01),when compared with values in NC virus treated cells.

The NC AAV or the Acat1 AAV (that include both siRNA Acat1 #54 and #55)were also injected into the hippocampal region of the AD mice at 4months of age. After a single bilateral injection, mice were allowed torecover. One month after injection, mice were sacrificed and the ACAT1enzyme activities in the mouse brain homogenates were analyzed by usinga standard ACAT enzyme activity assay in vitro. The result showed thatwhen compared with the control values, the Acat1 AAV reduced ACAT1enzyme activity by 42% (P<0.005).

Brain injections can cause various inflammatory responses in mice.Therefore, in a separate experiment, transcript levels of variousinflammatory markers were assessed one month after brain injections. Theresults showed that the brain injections of PBS and/or AAV causedalterations in the transcript levels of various inflammatory markers(iba, GFAP, TNFalpha, and iNOS); however, the degree of alteration wasmodest (i.e., within 20% of control values).

Subsequently, single bilateral injections of PBS, or NC AAV or Acat1 AAVwere made into the hippocampal region of AD mice with ACAT1(AD/ACAT1^(+/+)), or AD mice without ACAT1 (AD/ACAT1^(−/−)); both mousestrains were at 10 months of age. After injections, mice were allowed torecover, and were sacrificed two months later (at 12 months of age) todetermine Aβ1-42 content. The results showed that injecting AAVunexpectedly caused significant reduction of the Aβ 1-42 levels in theAD mice. Additional results also showed that injecting the AAV thatexpresses the Acat1 KD microRNA caused a clear reduction in the Aβ1-42level (see FIG. 1).

In the AD mouse brain, Acat1 genetic ablation (Acat1−/−) caused a 60-80%reduction in the Aβ1-42 content; however, residual Aβ1-42 was stillpresent in the brains of the AD/Acat1−/− mouse brain. In a controlexperiment, it was shown that, in the AD/Acat1−/− mouse brain, treatingwith either Acat1 AAV (FIG. 1) or with NC AAV (FIG. 1) caused about 25%reduction in the residual Aβ1-42 levels, confirming that injecting AAVcould cause significant Aβ1-42 reduction, in a manner independent of itsability to recognize the Acat1 mRNA sequence.

By using the residual A-beta 1-42 level remaining in the AD/Acat1^(−/−)mouse brain injected with AAV as the baseline value, it was estimatedthat the efficiency of Acat1 AVV to reduce Aβ1-42 in an ACAT1sequence-dependent manner was 70%.

Overall, these results showed that siRNAs against ACAT1 can be employedto cause inhibition of ACAT1 enzyme activity and to cause significantAβ1-42 reduction in the AD mouse brains in vivo, after cognitive deficitoccurred in these mice.

1. A method for decreasing the size and density of amyloid plaquescomprising administering to a subject in need of treatment an effectiveamount of an agent that selectively inhibits the activity ofAcyl-CoA:Cholesterol Acyltransferase 1 thereby decreasing the size anddensity of amyloid plaques in the subject.
 2. The method of claim 1,wherein the agent has an IC₅₀ value for Acyl-CoA:CholesterolAcyltransferase 2 which is at least twice the corresponding IC₅₀ valuefor Acyl-CoA:Cholesterol Acyltransferase
 1. 3. The method of claim 1,wherein the agent does not inhibit the expression ofAcyl-CoA:Cholesterol Acyltransferase
 2. 4. The method of claim 1,wherein the agent has an IC₅₀ value in the range of 1 nM to 100 μM. 5.The method of claim 1, wherein the agent is selectively delivered to thebrain of the subject.
 6. The method of claim 1, wherein the selectiveinhibitor of Acyl-CoA:Cholesterol Acyltransferase 1 is an siRNA ormicroRNA.
 7. The method of claim 6, wherein the Acyl-CoA:CholesterolAcyltransferase 1 inhibitor is administered via a liposome ornanoparticle.
 8. A method for decreasing cognitive decline associatedwith amyloid pathology comprising administering to a subject in need oftreatment an effective amount of an agent that selectively inhibits theactivity of Acyl-CoA:Cholesterol Acyltransferase 1 thereby decreasingcognitive decline associated with amyloid pathology in the subject. 9.The method of claim 8, wherein the agent has an IC₅₀ value forAcyl-CoA:Cholesterol Acyltransferase 2 which is at least twice thecorresponding IC₅₀ value for Acyl-CoA:Cholesterol Acyltransferase
 1. 10.The method of claim 8, wherein the agent does not inhibit the expressionof Acyl-CoA:Cholesterol Acyltransferase
 2. 11. The method of claim 8,wherein the agent has an IC₅₀ value in the range of 1 nM to 100 μM. 12.The method of claim 8, wherein the agent is selectively delivered to thebrain of the subject.
 13. The method of 8, wherein the selectiveinhibitor of Acyl-CoA:Cholesterol Acyltransferase 1 is an siRNA ormicroRNA.
 14. The method of claim 13, wherein the Acyl-CoA:CholesterolAcyltransferase 1 inhibitor is administered via a liposome ornanoparticle.
 15. A method for treating Alzheimer's Disease comprisingadministering to a subject in need of treatment an effective amount ofan agent that selectively inhibits the activity of Acyl-CoA:CholesterolAcyltransferase 1 thereby treating the subject's Alzheimer's Disease.16. The method of claim 15, wherein the agent has an IC₅₀ value forAcyl-CoA:Cholesterol Acyltransferase 2 which is at least twice thecorresponding IC₅₀ value for Acyl-CoA:Cholesterol Acyltransferase
 1. 17.The method of claim 15, wherein the agent does not inhibit theexpression of Acyl-CoA:Cholesterol Acyltransferase
 2. 18. The method ofclaim 15, wherein the agent has an 10₅₀ value in the range of 1 nM to100 μM.
 19. The method of claim 15, wherein the agent is selectivelydelivered to the brain of the subject.
 20. The method of claim 15,wherein the selective inhibitor of Acyl-CoA:Cholesterol Acyltransferase1 is an siRNA or microRNA.
 21. The method of claim 20, wherein theAcyl-CoA:Cholesterol Acyltransferase 1 inhibitor is administered via aliposome or nanoparticle.
 22. A method for decreasing the size anddensity of amyloid plaques comprising administering to a subject in needof treatment a microRNA that selectively inhibits the expression ofAcyl-CoA:Cholesterol Acyltransferase 1 thereby decreasing the size anddensity of amyloid plaques in the subject.
 23. The method of claim 22,wherein the microRNA is selectively delivered to the brain of thesubject.
 24. A method for decreasing cognitive decline associated withamyloid pathology comprising administering to a subject in need oftreatment a microRNA that selectively inhibits the expression ofAcyl-CoA:Cholesterol Acyltransferase 1 thereby decreasing cognitivedecline associated with amyloid pathology in the subject.
 25. The methodof claim 24, wherein the microRNA is selectively delivered to the brainof the subject.
 26. A method for treating Alzheimer's Disease comprisingadministering to a subject in need of treatment a microRNA thatselectively inhibits the expression of Acyl-CoA:CholesterolAcyltransferase 1 thereby treating the subject's Alzheimer's Disease.27. The method of claim 26, wherein the microRNA is selectivelydelivered to the brain of the subject.